Nanoparticles

ABSTRACT

Nanoparticle compositions comprising nanoparticles formed from π-conjugated cross-linked polymers are disclosed, together with their methods of manufacture and their applications. Owing to the nature of the cross-links formed therein, the nanoparticle compositions afford a high degree of manufacturing flexibility and control, as well as being amenable to facile purification for the purpose of imaging and electronics applications.

INTRODUCTION

The present invention relates to a nanoparticle composition comprisingnanoparticles formed from π-conjugated cross-linked polymer, as well asto their methods of manufacture and their uses.

BACKGROUND OF THE INVENTION

Photoluminescent conjugated polymer nanoparticles (CPNs) are currentlyviewed as attractive alternatives to Quantum Dots (QDs) for applicationsranging from biological imaging to consumer electronics.

QDs have previously shown promise in a number of in vitro and in vivobioimaging applications, where they can be internalized by cells,allowing individual organelles to be stained. However, their potentialfor in vivo oxidative degradation, which can release toxic heavy metalspecies (e.g. cadmium and lead), ultimately precludes their use inhumans or in long-term cell-tracking applications. Moreover, the use ofsuch heavy metals is heavily restricted in certain territories, therebyunderlining a need for less-toxic alternatives.

CPNs exhibit many of the desirable properties of QDs, including a smallsize (ca. 10-200 nm), photostable photoluminescence tunable across thevisible spectrum, and the ability to be isolated as stable dispersionsin water, whilst avoiding many of the toxicity-related drawbacks.

Behrendt et al. (Polym. Chem., 2013, 4, 1333-1336) discloses thatreplacing a proportion of the alkyl side chains present on polyfluorenenon-cross-linked co-polymers with a more hydrophilic side chain has asignificant influence on the size and optical properties of theresulting non-cross-linked CPNs.

Behrendt et al. (J. Mater. Chem. C, 2013, 1, 3297-3304) discloses hybridinorganic-organic composite nanoparticles formed from polyfluorenehaving pendant triethoxysilyl side chains that are cross-linkable underbasic conditions.

CN101323781A discloses nano-fluorescent microspheres having an outershell made from a water-soluble polymer and an inner shell being aconjugated fluorescent structure, and cross-links between the inner andouter shells.

Zhang et al (Gaofenzi Xuebao (2013), (4), 426-435) discloses thepreparation of chiral and fluorescent nanoparticles of hyperbranchedconjugated polymers by solvent chirality transfer technology.

In spite of the advances made to date, it is necessary that limitingfactors in the more widespread exploitation of phohotoluminescent CPNsbe addressed before they can realize their full potential as areplacement for QDs. Amongst these are improved production processes,greater manufacturing control, and superior purity for use in biologicalapplications.

The present invention was devised with the foregoing in mind.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided ananoparticle composition comprising a plurality of nanoparticles formedfrom a π-conjugated cross-linked polymer, the π-conjugated cross-linkedpolymer comprising

-   -   a) 80-99.9 mol. % of π-conjugated monomers, and    -   b) 0.1-20 mol. % of a cross-linker having the formula I shown        below:

wherein

Z₁ and Z₂ are monomeric moieties, and

Y is absent, a bond, or a linking group.

According to a second aspect of the present invention, there is provideda method of forming a nanoparticle composition defined herein, themethod comprising the step of forming the nanoparticles by emulsionpolymerisation, miniemulsion polymerisation or dispersion polymerisationtechniques to provide an aqueous suspension of nanoparticles.

According to a third aspect of the present invention, there is provideda nanoparticle composition obtainable, obtained, or directly obtained,by a method defined herein.

According to a fourth aspect of the present invention, there is provideda use of a nanoparticle composition defined herein in one or moreapplications defined herein.

According to a fifth aspect of the present invention, there is provideda nanoparticle dispersion comprising a nanoparticle composition asdefined herein dispersed throughout a dispersing medium.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the following meanings set out below.

References herein to the “Stille reaction” (also known as Stillecoupling) refer to a well-known chemical reaction coupling involving anorganotin compound with an sp²-hybridized organic halide catalyzed bypalladium. The reaction is widely used in organic synthesis. The use ofStille polymerisation reactions for the production of conjugated polymersystems is described in, for example, Chem. Rev. 2011, 111, 1493-1528.The general reaction scheme is shown below:

(R)₃Sn—R₁+X—R₂→R₁—R₂

whereinR is a hydrocarbyl substituent group, such as (1-6C)alkyl;R₁ and R₂ are both monomeric units to be coupled; and X is reactivegroup, typically a halide, such as CI, Br, I, or a pseudohalide, such asa triflate, CF₃SO₃ ⁻.

References to the “Suzuki reaction” refer to the well-known organicreaction of an aryl- or vinyl-boronic acid with an aryl- orvinyl-halide. Suzuki reactions are typically catalyzed by a palladium(0)complex catalyst. This reaction is well known in the chemical field andfollows the general reaction scheme shown below:

The reaction also works with pseudohalides, such as triflates (OTf),instead of halides. Boronic esters and organotrifluoroborate salts maybe used instead of boronic acids. For polymer synthesis, R₁ and R₂ willrepresent monomeric units.

The term “hydrocarbyl” includes both straight and branched chain alkyl,alkenyl and alkynyl groups.

The term “alkylene” includes both straight and branched chain alkylenegroups. References to individual alkylene groups such as “propylene” arespecific for the straight chain version only and references toindividual branched chain alkylene groups such as “isopropylene” arespecific for the branched chain version only. For example,“(1-20C)alkylene” includes (1-14C)alkylene, (1-12C)alkylene, propylene,isopropylene and t-butylene. A similar convention applies to otherradicals mentioned herein.

The terms “alkenylene” and “alkynylene” include both straight andbranched chain alkenyl and alkynyl groups.

The term “aryl” is used herein to denote phenyl, naphthalene oranthracene ring. In an embodiment, an “aryl” is phenyl or naphthalene,and particularly is phenyl.

The term “heteroaryl” or “heteroaromatic” means an aromatic mono-, bi-,or tri-cyclic ring incorporating one or more (for example 1-4,particularly 1, 2 or 3) heteroatoms (for example N, O, P, S, Si, Ge, Asor Se). Examples of heteroaryl groups are monocyclic, bicyclic andtricyclic groups containing from five to eighteen ring members. Theheteroaryl group can be, for example, a 5- or 6-membered monocyclicring, a 8-, 9-or 10-membered bicyclic ring or a 15-, 16-, 17- or18-membered tricyclic ring. Suitably each ring in a bicyclic ortricyclic ring system comprises five or six ring atoms.

The term “cross-linked” used herein in relation to polymers does notencompass linear or hyperbranched polymers. The polymeric “branches” ofhyperbranched polymers all emanate from a single focal point. Incontrast, the polymeric strands of the cross-linked polymers formingpart of the invention do not all converge to a single focal point.Rather, the strands of the cross-linked polymers forming part of theinvention are randomly cross-linked to one another throughout polymer,with none of the cross-linking sites representing a single focal pointin the sense of hyperbranched polymers. Furthermore, 4 or more polymericchains emanate from a given cross-linking site within the polymersforming part of the invention, whereas the single focal point (or otherbranch points) within a hyperbranched polymer is only 3 coordinate.Moreover, the cross-linked polymers forming part of the invention arecross-linked to the extent that they are insoluble in all solvents(including aqueous, organic, polar and non-polar solvents), whereashyperbranched polymers are commonly soluble.

Compositions of the Invention

As discussed hereinbefore, the present invention provides a nanoparticlecomposition comprising a plurality of nanoparticles formed from aπ-conjugated cross-linked polymer, the π-conjugated cross-linked polymercomprising

-   -   a) 80-99.9 mol. % of π-conjugated monomers, and    -   b) 0.1-20 mol. % of a cross-linker having the formula I shown        below:

wherein

Z₁ and Z₂ are monomeric moieties, and

Y is absent, a bond, or a linking group.

The nanoparticle compositions of the present invention offer a number ofadvantages when compared with the state of the art. Principally, thenanoparticles forming the present compositions are formed fromπ-conjugated cross-linked polymers. The π-conjugated cross-linkedpolymers themselves comprise a backbone of π-conjugated monomers, withcross-linking moieties interspersed along the π-conjugated backbone. Thestructure of the cross-linking moieties is such that one monomer spanstwo polymeric backbone chains. Therefore, during assembly of thepolymer, the incorporation of the cross-linking moieties into theπ-conjugated backbone chain provides a direct site for the propagationof a further π-conjugated backbone chain on both sides of thecross-linking moiety. Hence, the cross-links in the polymers forming thepresent nanoparticle compositions are formed in-situ during linking ofthe monomer units, meaning that the degree of cross-linking can bereadily adjusted simply by varying the concentration of cross-linker.Owing to their π-conjugated structures, cross-linked polymers of thistype provide good electron delocalisation properties. Such polymers alsooffer the possibility of electron delocalisation between adjacentbackbone chain via the cross-linker. In contrast to this direct, in-situformation of cross-links discussed above, prior art CPNs have focussedon the preparation of polymers formed from monomers havingspecially-modified pendant side chains that are amenable tocross-linking under certain conditions. Whilst being a viable method,such an approach necessarily requires the initial step of forming thepolymer backbone chains prior to placing the backbone chains undersuitable conditions to induce cross-linking between them. Thismulti-step approach is more complex than that used to prepare thepolymers forming the present compositions, and the degree ofcross-linking between the polymeric chains is notably more difficult tocontrol.

Aside from manufacturing simplicity and tuneability, the π-conjugatedcross-linked polymers forming part of the invention lend themselves toobtaining nanoparticle compositions exhibiting significantly higherlevels of purity. The insoluble cross-linker renders the nanoparticlecomposition insoluble in water and organic solvents, such that theπ-conjugated cross-linked polymers exhibit swelling when brought intocontact with a solvating solvent. Swelling the polymers in this mannerallows impurities trapped within the polymeric network, such ascatalysts and other reagents, to be easily removed by washing. Unlikeprior art compositions, the resulting high purity photoluminescentnanoparticle compositions are therefore highly suitable for use inbiological applications, such as bioimaging, and other in vivoprocesses.

Having regard to formula I, Z₁ is able to polymerise with π-conjugatedpolymer and aromatic monomers so as to form a first polymeric chain. Z₂is able to polymerise with π-conjugated polymer and aromatic monomers soas to form a second polymeric chain, adjacent to the first polymericchain, thereby linking together two adjacent polymeric chains.Accordingly, Z₁ and Z₂ may independently be selected from any of theexamples of the moieties forming part or all of the monomers that aredefined herein. In an embodiment, Z₁ and Z₂ are π-conjugated. In anotherembodiment, Z₁ and Z₂ are aromatic.

Still having regard to formula I, it will be appreciated that Z₁ and/orZ₂ may have more than 2 covalent attachment points (for attaching to theπ-conjugated monomers). For example Z₁ and/or Z₂ may have 3 covalentattachment points.

Still having regard to formula I, Y may be any suitable linker group,and may be π-conjugated or non-π-conjugated. Exemplary linker groupsinclude an atom (e.g. O, S), a metal (e.g. Ir, Pt, Rh, Re, Ru, Os, Cr,Cu, Pd, Au) or other group (e.g. —SiR₂—, —CH═CH—, —C₆H₄—). When Y is abond, it may be a single or double bond. When Y is absent, Z₁ isdirectly linked to Z₂, e.g. Z₁ is fused to Z₂ or is connected thereto bya common (shared) Spiro carbon atom.

The cross-linker of formula (I) may take a variety of forms. Inparticular, Y may be absent, a bond, or a linking group.

Where Y is absent (and Z₁ and Z₂ are linked directly to each other), thecross-linker may have a structure according for formula (Ia) below:

Examples of such cross-linkers include, but are not limited to:

In such embodiments, Z₁ may be directed connected to Z₂ in the sensethat Z₁ is fused to Z₂, or Z₁ and Z₂ share one or more common atoms.

Where Y is a bond (single or double), the cross-linker may have astructure according for formula (Ib) below:

Examples of such cross-linkers include, but are not limited to:

Where Y is a linking group, the linking group may be π-conjugated ornon-π-conjugated. Examples of cross-linkers having π-conjugated linkinggroups include, but are not limited to:

Examples of cross-linkers having non-π-conjugated linking groupsinclude, but are not limited to:

In certain embodiments, where Y is a linking group, the linking groupmay comprise additional monomeric moieties, Z_(n). In such embodiments,Y may have a structure according to formula (A) below:

wherein Y₁ is a linking group as defined herein;Z is a monomeric moiety and is as defined for Z₁ or Z₂ defined herein;andn is 1 or more (e.g. 1 or 2).In an embodiment, n is 1, and the cross-linker may have a structureaccording to formula (Ic) below:

Where Y₁ is a π-conjugated linking group, exemplary cross-linkers ofthis type include, but are not limited to:

Alternatively, where Y₁ is an atomic linking group, exemplarycross-linkers of this type include, but are not limited to:

Alternatively, the cross-linker of formula (Ic) may have a differentnumber of covalent attachment points (for attaching to the π-conjugatedmonomers). For example, the cross-linker may contain 5, 7, 8 or 9covalent attachment points, as illustrated below:

In another embodiment, each of monomeric moieties Z₁ and Z₂ may bebonded to Y by two separate bonds. Cross-linkers of this type may have astructure according to formula (Id) shown below:

In an embodiment, where Y is as defined in formula A, the cross-linkermay have a structure according to formula (Id′) below:

wherein Y₁ is a linking group as defined herein; andZ is a monomeric moiety and is as defined for Z₁ or Z₂ defined herein.Where Y₁ is an atomic linking group, exemplary cross-linkers of thistype include, but are not limited to:

Alternatively, the cross-linker of formula (Id′) may have a differentnumber of covalent attachment points (for attaching to the π-conjugatedmonomers). For example, the cross-linker may contain 4 (wherein Zcarries no covalent attachment points), 5, 7, 8 or 9 covalent attachmentpoints.

In an embodiment, Y is as defined in formula (A) and n is 2. In suchembodiments, the cross-linker may have a structure according to formula(Ie) below:

wherein Y₁ is a linking group as defined herein; andeach Z is independently a monomeric moiety and is as defined for Z₁ orZ₂ defined herein.Where Y₁ is a non-π-conjugated linking group, examples of suchcross-linkers include, but are not limited to:

In an embodiment, the nanoparticle composition comprises identicalcross-linkers, or a plurality of different cross-linkers.

In another embodiment, when Y is a linking group, said linking groupdoes not comprise additional monomeric moieties Z. In such embodiments,Z₁ and Z₂ are the only monomeric moieties present within thecross-linker.

In another embodiment, the cross-linker has the formula II shown below:

wherein

-   -   Y is absent, a bond, or a linking group.

In an embodiment, Y is absent, such that Z₁ is directly linked to Z₂,e.g. Z₁ is fused to Z₂ or is connected thereto by one or more common(shared) atoms (e.g. a spiro carbon atom). Suitably, Z₁ is connected toZ₂ by a common Spiro carbon atom.

In another embodiment, the cross-linker has the formula III shown below:

Suitably, the cross-linker has the following structure:

The nanoparticle composition comprises 80-99.9 mol. % of one or moreπ-conjugated monomers. Any suitable π-conjugated monomers capable ofpolymerising to form nanoparticles may be used.

In one embodiment, the π-conjugated polymers of the present invention donot comprise any carbon-carbon triple bonds. Thus, in one aspect, thepresent invention relates to π-conjugated cross-linked polymers that donot comprise any carbon-carbon triple bonds. The electrons in acarbon-carbon triple bond give rise to conformations in which theπ-electrons are not fully delocalised.

It will be appreciated by those skilled in the art that the monomericunits used to form the cross-linked π-conjugated polymers may comprise aselection of different chemical moieties that either alone or incombination provide a monomer having a π-conjugated ring system.

Examples of suitable π-conjugated ring systems that may be present inthe monomer units, either alone or in any suitable combination, includemono-cyclic aryl groups (e.g. phenyl rings), polycyclic aryl ringsystems (e.g. fluorene ring systems, naphthyl rings), mono-cyclicheteroaryl rings (e.g. thiophene rings) or polycyclic heteroaryl ringsystems (e.g. benzothiazole, benzodiazathazole rings,thieno[3,2-b]thiophene, or pyrrolo[3,4-c]pyrrole) or other conjugatedheterocyclic rings systems (e.g. pyrrolo-pyrrole-1,4-dione rings), andwherein each moiety is optionally substituted by one or more organicgroups, e.g. hydrocarbyl substituent groups optionally comprising 1 to30 carbon atoms and optionally comprising one or more heteroatoms (e.g.N, O, P, S, Si, Ge, As or Se), and, where two or more of such moietiesare present, they may be linked together by a bond or via an atomlinkage (e.g. such as in a bi-arylamine or tri-arylamine group).

Further examples of particular moieties that may form part or all of theπ-conjugated monomers include:

wherein R₃ and R₄ are each independently an organic substituent group(e.g. a hydrocarbyl substituent group optionally comprising 1 to 30carbon atoms and optionally comprising one or more heteroatoms (e.g. N,O, P, S, Si, Ge, As or Se), or an aromatic or heteroaromatic group);M is a metal (e.g. Ir, Pt, Rh, Re, Ru, Os, Cr, Cu, Pd, or Au);L is a ligand (e.g. a halide or a hydrocarbyl substituent groupoptionally comprising 1 to 30 carbon atoms and optionally comprising oneor more heteroatoms (e.g. N, O, S, Si, or P) or an aromatic orhetroaromatic group);and wherein each of the above structures is optionally furthersubstituted with one or more organic substituent groups (e.g. ahydrocarbyl substituent groups optionally comprising 1 to 30 carbonatoms and optionally comprising one or more heteroatoms (e.g. N, O, P,S, Si, Ge, As or Se) or an aromatic or heteroaromatic group).

In an embodiment, the π-conjugated monomers each independently comprisea moiety having the formula IV shown below:

-   -   wherein    -   R₁ and R₂ are each independently a group:

—X-Q

-   -   -   wherein        -   X is selected from the group consisting of (1-30C)alkylene,            (2-30C)alkenylene, (2-30C)alkynylene, —[(CH₂)₂—O]_(n)—,            —[O—(CH₂)₂]_(n)— and —[O—Si(R_(z))₂]_(n) (wherein R_(z) is            (1-4C)alkyl and n is 1 to 30), and        -   Q is a terminal group selected from hydrogen, methyl,            hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —C═CH₂,            —C≡CH, —SH, -biotin, -streptavidin and a polymerisable group            selected from acrylates, epoxy and styrene,

    -   or R₁ and R₂ are linked so that, together with the carbon atom        to which they are attached, they form a ring.

In another embodiment, π-conjugated monomers each independently have astructure defined by formula V shown below:

-   -   wherein    -   R₁ and R₂ are as defined hereinbefore;    -   A₁ and A₂ are independently absent or selected from any one of        the following moieties:

-   -   -   and wherein R₃ and R₄ are each independently a group:

—X′-Q′

-   -   -   -   wherein            -   X¹ is selected from the group consisting of                (1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene,                —[(CH₂)₂—O]_(n)—, —[O—(CH₂)₂]_(n)—, and                —[O—Si(R_(x))₂]_(n)— (wherein R_(z) is (1-4C)alkyl and n                is 1 to 30),            -   Q¹ is a terminal group selected from hydrogen, methyl,                hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —C═CH₂,                —C≡CH, —SH, -biotin, -streptavidin, and a polymerisable                group selected from acrylates, epoxy or styrene;            -   M is a metal selected from Ir, Pt, Rh, Re, Ru, Os, Cr,                Cu, Pd and Au;            -   L is a ligand independently selected from the group                consisting of halo, (1-30C)hydrocarbyl optionally                comprising one or more heteroatoms selected from N, O,                S, Si, Ge, As or P, or an aryl or heteroaryl group                optionally substituted with one or more substituents                selected from (1-4C)alkyl, halo, aryl or heteroaryl; and            -   p is 1 to 4.

In another embodiment, the π-conjugated monomers each independently havea structure defined by formula VI below:

R₁, R₂, A₁ and A₂ are as defined hereinbefore.

In another embodiment, A₁ and A₂ are independently absent or selectedfrom any one of the following moieties:

wherein R₃, R₄, M, L and p are as defined hereinbefore.

In another embodiment both A₁ and A₂ are absent.

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene, —[(CH₂)₂—O]_(n)—,—[O—(CH₂)₂]_(n)— and —[O—Si(R_(z))₂]_(n)— (wherein R_(z) is methyl and nis 1 to 30);Q and Q¹ are independently a terminal group selected from hydrogen,methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —C═CH₂, —C≡CHand a polymerisable group selected from acrylates, epoxy and styrene;M is a metal selected from Ir, Pt, Rh, Re, Ru, Os, Cr, Cu, Pd and Au;L is a ligand independently selected from the group consisting of halo,(1-30C)hydrocarbyl optionally comprising one or more heteroatomsselected from N, O, S, Si or P, or an aryl or heteroaryl groupoptionally substituted with one or more substituents selected from(1-4C)alkyl, halo, aryl or heteroaryl; andp is 1 to 4

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(1-20C)alkylene, (2-20C)alkenylene, (2-20C)alkynylene, —[(CH₂)₂—O]_(n)—and —[O—(CH₂)₂]_(n)— (wherein n is 1 to 20);Q and Q¹ are independently a terminal group selected from hydrogen,methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl, amino, —C═CH₂ and—C≡CH.M is a metal selected from Ir, Pt, Cr, Cu, Pd and Au;L is a ligand independently selected from the group consisting of halo,(1-20C)hydrocarbyl optionally comprising one or more heteroatomsselected from N, O, or S, or an aryl or heteroaryl group optionallysubstituted with one or more substituents selected from (1-4C)alkyl,halo, aryl or heteroaryl; andp is 1 to 4.

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(1-20C)alkylene, —[(CH₂)₂—O]_(n)— and —[O—(CH₂)₂]_(n)— (wherein n is 1to 20);Q and Q¹ are independently a terminal group selected from hydrogen,methyl, hydroxyl, carboxy, (1-4C)alkoxycarbonyl and amino;M is a metal selected from Ir, Pt, Cr, Cu, Pd and Au;L is a ligand independently selected from the group consisting of arylor heteroaryl, optionally substituted with one or more substituentsselected from (1-4C)alkyl, halo, aryl or heteroaryl; andp is 1 to 4.

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(1-20C)alkylene, —[(CH₂)₂—O]_(n)— and —[O—(CH₂)₂]_(n)— (wherein n is 1to 20);Q and Q¹ are independently a terminal group selected from hydrogen,methyl, (1-2C)alkoxycarbonyl and hydroxyl;

M is Ir;

L is a ligand independently selected from the group consisting of arylor heteroaryl, optionally substituted with one or more substituentsselected from aryl or heteroaryl; andp is 1 to 2.

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(4-12C)alkylene, —[(CH₂)₂—O]_(n)— and —[O—(CH₂)₂]_(n)— (wherein n is 1to 15);Q and Q¹ are independently a terminal group selected from hydrogen,methyl, (1-2C)alkoxycarbonyl and hydroxyl;

M is Ir;

L is a ligand independently selected from the group consisting of phenylor 6-membered heteroaryl, optionally substituted with one or moresubstituents selected from phenyl or 6-membered heteroaryl; andp is 1 to 2.

In another embodiment, when present:

X and X¹ are independently selected from the group consisting of(4-12C)alkylene and —[(CH₂)₂—O]_(n)— (wherein n is 1 to 15);Q and Q¹ are independently a terminal group selected from hydrogen,(1-2C)alkoxycarbonyl and methyl;

M is Ir;

L is a ligand independently selected from the group consisting of phenylor 6-membered heteroaryl, optionally substituted with one or moresubstituents selected from phenyl or 6-membered heteroaryl; andp is 1 to 2

In any of the embodiments mentioned hereinbefore, X and/or X¹ may alsobe —(CH₂)_(m)(CF₂)_(n)— (wherein m is 0 to 30 and n is 1 to 30) and Qand/or Q¹ may also be —CF₃.

In another embodiment, the π-conjugated monomers are each independentlyselected from any of the following structures:

In another embodiment, the π-conjugated monomers are each independentlyselected from any of the following structures:

In another embodiment, the nanoparticle composition is an aqueoussuspension. The aqueous medium provides a water-based vehicle in whichthe nanoparticles are dispersed. The medium may comprise additionalcomponents, such as dissolved materials and other water-misciblesolvents. Suitably, the aqueous medium is water. More suitably, theaqueous medium is purified water.

In another embodiment, the nanoparticles forming the nanoparticlecomposition have a particle size (Z-average, measured by DLS) of 20-400nm. Suitably, the nanoparticles forming the nanoparticle compositionhave a particle size of 30-400 nm. More suitably, the nanoparticles havea particle size of less than 30-300 nm. Even more suitably, thenanoparticles have a particle size of less than 30-250 nm. Even moresuitably, the nanoparticles have a particle size of less than 30-200 nm.Most suitably, the nanoparticles have a particle size of less than30-100 nm.

In another embodiment, the nanoparticles forming the nanoparticlecomposition have a particle size of 20-400 nm. More suitably, thenanoparticles have a particle size of less than 20-300 nm. Even moresuitably, the nanoparticles have a particle size of less than 20-250 nm.Even more suitably, the nanoparticles have a particle size of less than20-200 nm. Most suitably, the nanoparticles have a particle size of lessthan 20-100 nm.

In another embodiment, the nanoparticles forming the nanoparticlecomposition have a particle size of 10-400 nm. More suitably, thenanoparticles have a particle size of less than 10-300 nm. Even moresuitably, the nanoparticles have a particle size of less than 10-250 nm.Even more suitably, the nanoparticles have a particle size of less than10-200 nm. Most suitably, the nanoparticles have a particle size of lessthan 10-100 nm.

In another embodiment, the polymers forming part of the presentinvention have a degree of polymerisation of 10 to 800, more suitably 20to 600.

In another embodiment, the nanoparticle composition comprises 1-10 mol %of the cross linker. Suitably, the nanoparticle composition comprises2-8 mol % of the cross linker. More suitably, the nanoparticlecomposition comprises 3-7 mol % of the cross linker. Most suitably, thenanoparticle composition comprises 4.5-5.5 mol % of the cross linker.

In another embodiment, the nanoparticle composition of the invention mayfurther comprise a stabiliser to maintain the particles in suspension.Any suitable stabiliser may be used such as, for example, non-ionic,cationic or anionic stabilisers known in the art. Particular examples ofsuitable stabilisers include non-ionic stabilisers, for example: TritonX series octylphenol ethoxylates, Tergitol series nonylphenolethoxylates (Dow Chemical Company); Brij series poly(oxyethylene) glycolalkyl ethers, Superonic series, Tween series polysorbate surfactants(Croda); Pluronic series of block copolymers based on ethylene oxide andpropylene oxide (BASF); Tetronic series tetra functional blockcopolymers based on ethylene oxide and propylene oxide, Lutensol series(BASF); Igepal series Rhodasurf series and Antarox series (Rhodia); andMerpol series (Stepan Co.)

In another embodiment, the nanoparticle composition further comprises ananionic stabiliser, for example sodium dodecylsulphate (SDS), and/or acationic stabiliser, for example cetyl trimethylammonium bromide (CTAB).

Dispersions of the Invention

As discussed hereinbefore, the present invention also provides ananoparticle dispersion comprising a nanoparticle composition as definedherein dispersed throughout a dispersing medium.

In an embodiment, the dispersing medium is a liquid (e.g. water or asolution of monomers). Aqueous dispersing media may be particularlysuitable where the dispersion is intended for biological applications.

Alternatively, the dispersing medium may be a solid (e.g. a polymericmatrix). Dispersions where the dispersing medium is a polymeric matrixmay be particularly suitable for use as LED phosphors.

In another embodiment, the nanoparticle dispersions are prepared suchthat the loading of nanoparticle composition is high. Suitably, theconcentration of the nanoparticles in the dispersing medium is greaterthan or equal to 15 mM. More suitably, the concentration of thenanoparticles in the dispersing medium is greater than or equal to 20mM. Suitably, the concentration of the nanoparticles in the dispersingmedium is greater than or equal to 25 mM. The aforementionedconcentrations are based on the initial monomer concentrations used inthe polymerisation reaction, and assumes 100% conversion of the monomersto the polymer.

Alternatively, depending on the application of interest, thenanoparticle dispersion may be more dilute. In an embodiment, theconcentration of the nanoparticles in the dispersing medium (e.g. water)is less than or equal to 15 mg/ml. Such dispersions may be particularlyuseful in biological applications.

In an alternative embodiment, the concentration of the nanoparticles inthe dispersing medium (e.g. a polymeric matrix) is less than or equal to5 wt %. Suitably, the concentration of the nanoparticles in thedispersing medium is less than or equal to 3 wt %. More suitably, theconcentration of the nanoparticles in the dispersing medium is less thanor equal to 1 wt %. Such dispersions may find application where thenanoparticles are being used as LED phosphors.

Methods of the Invention

As discussed hereinbefore, the present invention also provides a methodof forming a nanoparticle composition defined herein, the methodcomprising the step of forming the nanoparticles by emulsionpolymerisation, miniemulsion polymerisation or dispersion polymerisationtechniques to provide an aqueous suspension of nanoparticles.

Emulsion polymerisation, miniemulsion polymerisation and dispersionpolymerisation techniques will be known to one of skill in the art.

In the case of emulsion polymerisation, the monomeric components aredissolved in a suitable organic solvent (e.g. chlorobenzene, toluene orxylenes) along with the catalyst (e.g. Pd(PPh₃)₄, IPr*PdTEACl₂ orPd₂(dba)₃/P(o-tol)₃). This solution is then added to an aqueous mediumof water, tetraethylammonium hydroxide solution (40% in water) and asuitable emulsifier. Any suitable emulsifier may be used, such as, forexample, SDS, Triton X102, Brij L23, and/or Tween 20. The resultantemulsion may be stirred and/or ultrasonicated to form an emulsion,suitably a mini-emulsion. The emulsion mixture may then be gently heatedto a temperature of between 30 and 100° C. (for Pd(PPh₃)₄,Pd₂(dba)₃/P(o-tol)₃ suitably between 70 and 95° C., and more suitablybetween 80 and 95° C.; and for IPr*PdTEACl₂ ideally 30° C.) for periodof time (e.g. from 1 hour to 2 days) to form the polymericnanoparticles. A person skilled in the art will appreciate that thetemperature of heating depends on catalyst system employed (as per theexample section herein).

In an embodiment, the nanoparticles are formed by Suzuki coupling orStille coupling reactions. Such coupling reactions are known in the art.

In another embodiment, the nanoparticles are formed by reacting7-monomeric moieties as defined herein with a pre-made cross-linkingmoiety as defined herein.

In another embodiment, the method further comprises the step ofpurifying the aqueous suspension of nanoparticles. Suitably, the aqueoussuspension of nanoparticles is purified by contacting the aqueoussuspension of nanoparticles with at least one organic solvent.

In another embodiment, contacting the aqueous suspension ofnanoparticles with at least one suitable organic solvent causesprecipitation of the nanoparticles. The precipitated nanoparticles maythen be centrifuged, with the supernatant then decanted to leave thepurified nanoparticles. Optionally, the purified nanoparticle may beresuspended in water, and the purification process then repeated.

In another embodiment, when the nanoparticles are lipophilic, the atleast one organic solvent is a polar solvent that is miscible with water(e.g. methanol).

In another embodiment, when the nanoparticles are hydrophilic, the atleast one organic solvent is a non-polar solvent.

Uses of the Nanoparticle Compositions

As discussed hereinbefore, the present invention also provides a use ofa nanoparticle composition defined herein in one or more applicationsselected from the group consisting of biological or non-biologicalimaging or sensing, down-conversion of LED light, anti-counterfeitencoding, displays, cell-sorting/flow cytometry, long-term celltracking, and flow visualisation.

In an embodiment, the nanoparticle composition is used in in vivo or invitro imaging or sensing applications.

EXAMPLES

Examples of the invention will now be described, for the purpose ofreference and illustration only, with reference to the accompanyingfigures, in which:

FIG. 1 shows DLS particle size histograms of the cross-linkednanoparticles of Example 1 in water (solid line) or THF (broken line).

FIG. 2 shows UV/Vis spectra of the cross-linked nanoparticles of Example1 in water (solid line) or THF (broken line).

FIG. 3 shows PL spectra of the cross-linked nanoparticles of Example 1in water (solid line) or THF (broken line).

FIG. 4 shows DLS particle size histograms of the cross-linkednanoparticles of Example 2 in water (solid line) and THF (broken line)dispersants.

FIG. 5 shows UV/Vis (broken line) and PL (solid line) spectra of thecross-linked nanoparticles of Example 2.

FIG. 6 shows DLS sizing histograms of cross-linked phosphorescentnanoparticles in water (solid line) or THF (broken line) of thecross-linked nanoparticles of Example 3.

FIG. 7 shows UV/Vis spectra of the cross-linked nanoparticles of Example3 in water (solid line) or THF (broken line).

FIG. 8 shows PL spectra of the cross-linked nanoparticles of Example 3in water (solid line) or THF (broken line).

FIG. 9 shows DLS sizing histograms of the cross-linked nanoparticles ofExample 4 in water (solid line) and THF (broken line).

FIG. 10 shows DLS sizing histograms of the cross-linked nanoparticles ofExample 5 in water.

FIG. 11 shows DLS sizing histograms of the cross-linked nanoparticles ofExample 6 in water (broken line) and THF (solid line).

FIG. 12 shows absorption and emission spectra of the cross-linkednanoparticles of Examples 4 (FIG. 12a ), 5 (FIG. 12b ) and 6 (FIG. 12c).

Example 1—Cross-Linked PFO Nanoparticles Synthesis

Referring to Scheme 1 and Table 1 shown below, sodium dodecyl sulphate(SDS) (50.0 mg) and deionised water (10 mL) were transferred to aSchlenk tube and the resultant solution was degassed by bubbling withargon for 20 minutes. Monomer A (see Table 1), crosslinker B (seeTable 1) and monomer C (58.6 mg, 9.12×10⁻² mmol) were dissolved intoluene (1 mL), to which hexadecane (78 μL) was also added, and thissolution was degassed for 5 minutes in the same manner.Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10⁻³ mmol) wasadded to the monomer solution, which was then transferred to thereaction vessel. The reaction mixture was emulsified by ultrasonication(Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for2 minutes while cooling with an ice bath. The Schlenk tube was resealedand the miniemulsion was heated to 72° C., followed by addition of 1Maqueous sodium hydroxide solution (365 μL), and the reaction mixture wasstirred for 16 hours. After cooling to room temperature, the cap of thereaction vessel was removed and the emulsion was stirred for 5 hours toremove the residual toluene.

TABLE 1 Reaction variables for synthesis of cross-linked PFOnanoparticles Sample Monomer A Crosslinker B Name (mass, moles) (mass,moles) NP-X2.5 45.0 mg 2.9 mg 8.21 × 10⁻² mmol 4.6 × 10⁻³ mmol NP-X540.0 mg 5.8 mg 7.29 × 10⁻² mmol 9.1 × 10⁻³ mmol NP-X10 30.0 mg 11.6 mg5.47 × 10⁻² mmol 1.82 × 10⁻² mmol

Surfactant Removal and DLS Analysis (Nanoparticles in Water)

A 400 μL aliquot of the crude nanoparticle suspension was diluted with1.6 mL of deionised water, to which Amberlite XAD-2 resin (20 mg,pre-washed with 2×2 mL of water) was added. The suspension was shaken atroom temperature for 15 minutes before decanting off the nanoparticlesuspension. This Amberlite XAD-2 purification step was repeated, afterwhich time the suspension no longer foamed upon shaking and was filteredthrough glass wool prior to dynamic light scattering (DLS) analysis ofparticle size using a Malvern Zetasizer Nano ZS. Results are shown inTable 2 and FIG. 1.

TABLE 2 DLS analysis of cross-linked PFO nanoparticles in water Z- Sizeby St. Sample Average Intensity Dev. Name (d · nm) (d · nm) (nm) PdINP-X2.5 128 154 69 0.16 NP-X5 130 151 60 0.14 NP-X10 129 150 56 0.13

DLS Analysis (Nanoparticles in THF)

A 200 μL aliquot of the crude nanoparticle suspension was flocculatedthrough addition of 1.3 mL toluene and the polymer was isolated bycentrifugation (14,000 rpm, 1 minute) and decantation of thesupernatant. The polymer was dried in air to remove residual methanolbefore dissolving in tetrahydrofuran (THF, 1 mL). The resultantsuspension was measured directly using a Malvern Zetasizer Nano ZS.Results are shown in Table 3 and FIG. 1.

TABLE 3 DLS analysis of cross-linked PFO nanoparticles in THF SampleZ-Average Size by Intensity St. Dev. name (d · nm) (d · nm) (nm) PdINP-X2.5 — — — n/a^([a]) NP-X5 174  198 (99.6%)  74 (99.6%) 0.13 4827(0.4%)^([b]) 711 (0.4%)^([a]) NP-X10 147 175 73 0.15 ^([a])secondarypeak likely to result from a small proportion of aggregatednanoparticles

UV/Vis Analysis (Nanoparticles in Water or THF)

Following surfactant removal via treatment with Amberlite XAD-2, 40 μLof the nanoparticle suspension was diluted with 3 mL of water. UV-Visabsorption spectra of the nanoparticles at this concentration wererecorded on a Varian Cary 55 5000UV-Vis-NIR spectrophotometer at roomtemperature. FIG. 2 shows UV/Vis spectra of the cross-linked PFOnanoparticles.

Photoluminescence (PL) Analysis (Nanoparticles in Water or THF)

Following surfactant removal via treatment with amberlite XAD-2, 40 μLof the nanoparticle suspension was diluted with 3 mL of water. PLspectra were recorded on a Varian Cary Eclipse fluorimeter. FIG. 2 showsPL spectra of the cross-linked PFO nanoparticles

Photoluminescence (PL) Analysis (Nanoparticles in Water)

Photoluminescencemeasurements were obtained using a Fluoromax-4spectrofluorometer. Measurements were carried out on dilute dispersionsof the nanoparticles in water (800 μL, abs >1), using the same volume ofwater for background measurements. The results are provided in Table 4.

TABLE 4 Optical properties of PFO nanoparticles in water Sample Nameλ_(max) λ_(em) ^([a]) NP-X2.5 390 440 NP-X5 390 438 NP-X10 390 437^([a])λ_(ex) = 380 nm

Example 2—Ethyl Ester-Functionalised Cross-Linked PFO NanoparticlesSynthesis

Referring to Scheme 2 shown below, sodium dodecyl sulfate (50.0 mg) anddeionised water (10 mL) were transferred to a Schlenk tube and theresultant solution was degassed by bubbling with argon for 20 minutes.Crosslinker A (5.8 mg, 9.12×10⁻³ mmol), monomer B (44.4 mg, 7.30×10⁻²mmol) and monomer C (58.6 mg, 9.12×10⁻² mmol) were dissolved in toluene(1 mL), to which hexadecane (78 μL) was also added, and this solutionwas degassed for 5 minutes in the same manner.Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10⁻³ mmol) wasadded to the monomer solution, which was then transferred to thereaction vessel. The reaction mixture was emulsified by ultrasonication(Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for2 minutes while cooling with an ice bath. The Schlenk tube was resealedand the miniemulsion was heated to 72° C., followed by addition of 1Maqueous sodium hydroxide solution (365 μL), and the reaction mixture wasstirred for 16 hours. After cooling to room temperature, the cap of thereaction vessel was removed and the emulsion was stirred for 5 hours toremove the residual toluene.

DLS Analysis (Nanoparticles in Water or THF)

Surfactant removal was carried out using the general procedure describedin Example 1. Flocculation and resuspension in THF were carried outusing the general procedure described in Example 1. DLS analysis wascarried out as in Example 1, using either water or THF as thedispersant. The results are provided in Table 5 and FIG. 4.

TABLE 5 DLS analysis of ethyl ester-functionalised nanoparticles inwater or THF Sample Z-Average Size by Intensity St. Dev Name Dispersant(d · nm) (d · nm) (nm) PdI NP-X5E40 Water 118 139 56 0.14 NP-X5E40 THF170 204 82 0.16

UV/Vis and PL Analysis (Nanoparticles in Water)

The general UV/Vis and PL analytical procedures described in Example 1were used to record the UV/Vis and PL spectra of the nanoparticles indilute aqueous dispersion. The results are provided in FIG. 5.

PLanalysis (Nanoparticles in Water)

PL measurements were obtained using the general method described inExample 1. The results are provided in Table 6.

TABLE 6 Optical properties of ethyl ester- functionalised nanoparticlesin water Sample Name λ_(max) λ_(em) ^([a]) NP-X5E40 391 432 ^([a])λ_(ex)= 380 nm

Example 3—Cross-Linked Phosphorescent Nanoparticles Method

Referring to Scheme 3 and Table 7 shown below, sodium dodecyl sulfate(50.0 mg) and deionised water (10 mL) were transferred to a Schlenk tubeand the resultant solution was degassed by bubbling with argon for 20minutes. Monomers A (see Table 7), C (20.5 mg, 1.82×10⁻² mmol) and D(58.6 mg, 9.12×10⁻² mmol) and crosslinker B (5.8 mg, 9.12×10⁻³ mmol)were dissolved in toluene (1 mL), to which hexadecane (78 μL) was alsoadded, and this solution was degassed for 5 minutes in the same manner.Tetrakis(triphenylphosphine)palladium(0) (2.2 mg, 9.13×10⁻³ mmol) wasadded to the monomer solution, which was then transferred to thereaction vessel. The reaction mixture was emulsified by ultrasonication(Cole Parmer 750W ultasonicator, fitted with microtip, on 22% power) for2 minutes while cooling with an ice bath. The Schlenk tube was resealedand the miniemulsion was heated to 72° C., followed by addition of 1Maqueous sodium hydroxide solution (365 μL), and the reaction mixture wasstirred for 16 hours. After cooling to room temperature, the cap of thereaction vessel was removed and the emulsion was stirred for 5 hours toremove the residual toluene.

TABLE 7 Reaction variables for synthesis of cross- linked phosphorescentnanoparticles Sample Monomer A Monomer A Name Side Chain (R¹) (mass,moles) NP-XIr1 Octyl 30.0 mg NP-XIr2 MeO-PEG3 5.47 × 10⁻² mmol 33.7 mg5.57 × 10⁻² mmol

DLS Analysis (Nanoparticles in Water or THF)

Surfactant removal was carried out using the general procedure describedin Example 1. Flocculation and resuspension in THF were carried outusing the general procedure described in Example 1. DLS analysis wascarried out as in Example 1, using either water or THF as thedispersant. The results are provided in Table 8 and FIG. 6.

TABLE 8 DLS analysis of cross-linked phosphorescent nanoparticles inwater or THF Size by Sample Z-Average Intensity St. Dev Name Dispersant(d · nm) (d · nm) (nm) PdI NP-XIr1 Water 131 158  69 0.15 NP-XIr1 THF167 210 109 0.18 NP-XIr2 Water 126  150 (99.3%)  70 (99.3%) 0.19 4709(0.7%)^([a]) 774 (0.7%)^([a]) NP-XIr2 THF 165 205 98 0.18^([a])Secondary peak likely to result from a small proportion ofaggregated nanoparticles

UV/Vis and PL Analysis (Nanoparticles in Water or THF)

The general UV/Vis and PL analytical procedures described in Example 1were used to record the UV/Vis (FIG. 7) and PL (FIG. 8) spectra of thenanoparticles in dilute aqueous dispersion or THF.

PL Analysis (Nanoparticles in Water)

PLmeasurements were obtained using the general method described inExample 1. The results are provided in Table 9.

TABLE 9 Optical properties of cross-linked phosphorescent nanoparticlesin water Sample Name λ_(max) λ_(em) ^([a]) NP-Ir1 392 609 NP-Ir2 392 609^([a])λ_(ex) = 390 nm

Example 4—PEG3 Functionalised 10% Cross-Linked PFO NanoparticlesSynthesis

Referring to Scheme 4 shown below, tetraethylammonium hydroxide solution(40% in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution(50 ml) of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % inde-ionised water) in a 100 ml three necked round bottom flask. Thencontents were then through degassed for 30 mins by bubbling nitrogen gasthrough the stirred solution. Then a separate 10 ml two necked roundbottom flask was used to mix together the monomers in the organicsolvent prior to addition to the reaction flask.9,9-dioctylfluorene-2,7-di-boronic acid-bis(1,3-propanediol)ester(0.1151 g, 0.2 mmol),2,7-dibromo-9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorene (0.0967g, 0.16 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (0.0126 g,0.02 mmol) were dissolved in xylene (2 ml). The monomer solution wasdegassed and then the catalyst IPr*PdTEACl₂ (0.0095 g, 0.008 mmol) wasadded, followed by further degassing of the resultant solution. Asyringe was used to transfer the monomer/catalyst into the stirredsurfactant/base solution in the main reaction flask now maintained at30° C. with stirring and maintaining under nitrogen gas for 24 h.

DLS Analysis (Nanoparticles in Water or THF)

500 μl of sample was transferred to centrifuge vial the 1.5 ml ofmethanol was added. The sample vial was centrifuged at 14,000 rpm for 5min then the liquid was decanted. Crude sample was washed with methanol3 times and re-dispersed in THF in order to measure the size ofparticles. Neat products without further purification were alsoinvestigated. The results are shown in FIG. 9 and Table 10.Concentrations of polymer in water was 23 μg/ml.

TABLE 10 Particle sizes of CPNs in water and THF at 25° C. Size Dz STDSample (nm) (nm) (nm) PdI LM55 Neat 50 44 26.81 0.244 LM55 in THF 108218 51.80 0.217

Optical Properties

Referring to Table 11 and FIG. 12, LM55 exhibited maxima band at 370 nmbut no β-phase was observed.

TABLE 11 Summarized optical properties of cross-linked polymer in waterFinal polymer Size λ_(abs) λ_(em) Sample conc. (mg/ml) (nm) (nm) (nm)E_(g)* LM55 2.5 50 370 420, 441 2.91

Example 5—PEG3 Functionalised 5% Cross-Linked PFO NanoparticlesSynthesis

Referring to Scheme 5 shown below, tetraethylammonium hydroxide solution(40% in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution(50 ml) of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % inde-ionised water) in a 100 ml three necked round bottom flask. Thencontents were then through degassed for 30 mins by bubbling nitrogen gasthrough the stirred solution. Then a separate 10 ml two necked roundbottom flask was used to mix together the monomers in the organicsolvent prior to addition to the reaction flask.9,9-dioctylfluorene-2,7-di-boronic acid-bis(1,3-propanediol)ester(0.1151 g, 0.2 mmol), 2,7-dibromo-9,9-dioctylfluorene (0.0768 g, 0.14mmol), 2,7-dibromo-9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorene(0.0242 g, 0.04 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene(0.0063 g, 0.01 mmol) were dissolved in xylene (2 ml). The monomersolution was degassed and then the catalyst IPr*PdTEACl₂ (0.0095 g,0.008 mmol) was added, followed by further degassing of the resultantsolution. A syringe was used to transfer the monomer/catalyst into thestirred surfactant/base solution in the main reaction flask nowmaintained at 30° C. with stirring and maintaining under nitrogen gasfor 24 h.

DLS Analysis (Nanoparticles in Water or THF)

500 μl of sample was transferred to centrifuge vial the 1.5 ml ofmethanol was added. The sample vial was centrifuged at 14,000 rpm for 5min then the liquid was decanted. Crude sample was washed with methanol3 times and re-dispersed in THF in order to measure the size ofparticles. Neat products without further purification were alsoinvestigated. The results are shown in FIG. 10 and Table 12.Concentrations of polymer in water was 23 μg/ml.

TABLE 12 Particle sizes of CPNs in water at 25° C. Size Dz STD Sample(nm) (nm) (nm) PdI LM56 Neat 55 41 26.23 0.381

Optical Properties

Referring to Table 13 and FIG. 12, LM56 showed absorption peak at 378nm.

TABLE 13 Summarized optical properties of cross-linked polymer in waterFinal polymer Size λ_(abs) λ_(em) Sample conc. (mg/ml) (nm) (nm) (nm)E_(g)* LM56 2.5 55 378, 435 421, 436, 453 2.78

Example 6—PEG12 Functionalised 10% Cross-Linked PFO NanoparticlesSynthesis

Referring to Scheme 6 below, tetraethylammonium hydroxide solution (40%in water) (0.1567 g, 0.4 mmol), was added to an aqueous solution (50 ml)of non-ionic surfactant, Triton x-102 (2.5 g, 5 wt % in de-ionisedwater) in a 100 ml three necked round bottom flask. Then contents werethen through degassed for 30 mins by bubbling nitrogen gas through thestirred solution. Then a separate 10 ml two necked round bottom flaskwas used to mix together the monomers in the organic solvent prior toaddition to the reaction flask. 9,9-dioctylfluorene-2,7-di-boronicacid-bis(1,3-propanediol)ester (0.1151 g, 0.2 mmol),2,7-dibromo-9,9-bis(polyethylene glycol monoether)fluorene (0.2255 g,0.16 mmol) and 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (0.0126 g, 0.02mmol) were dissolved in xylene (2 ml). The monomer solution was degassedand then the catalyst IPr*PdTEACl₂ (0.0095 g, 0.008 mmol) was added,followed by further degassing of the resultant solution. A syringe wasused to transfer the monomer/catalyst into the stirred surfactant/basesolution in the main reaction flask now maintained at 30° C. withstirring and maintaining under nitrogen gas for 24 h.

DLS Analysis (Nanoparticles in Water or THF)

500 μl of sample was transferred to centrifuge vial the 1.5 ml ofmethanol was added. The sample vial was centrifuged at 14,000 rpm for 5min then the liquid was decanted. Crude sample was washed with methanol3 times and re-dispersed in THF in order to measure the size ofparticles. Neat products without further purification were alsoinvestigated. The results are shown in FIG. 11 and Table 14.Concentrations of polymer in water was 23 μg/ml.

TABLE 14 Particle sizes of CPNs in water and THF at 25° C. Size Dz STDSample (nm) (nm) (nm) PdI LM02-6 Neat 244 13 103.2 0.359 LM02-6 in THF74 847 10.97 0.489

Optical Properties

Table 15 and FIG. 12 show summarized optical properties for LM02-6 inwater.

TABLE 15 Summarized optical properties of cross-linked polymer in waterFinal polymer Size λ_(abs) λ_(em) Sample conc. (mg/ml) (nm) (nm) (nm)E_(g)* LM02-6 2.5 244 N/A 419, 441 N/A

Example 7-5% 1,3-Diphenoxypropane Cross-Linked PolyfluoreneNanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmol),1,3-bis(3,5-dibromophenoxy)propane (10.9 mg, 20 μmol),tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmop. The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkylight green solution. DLS (water): Z-average=110 nm, Pdl=0.156, D_(n)=69nm and SD=21.0 nm. UV-Vis Abs. (water): λ_(max)=379 nm, λ_(sec.)=432 nm,λ_(onset)=455 nm. UV-Vis PL (water): λ_(max)=439 nm, λ_(sec.)=467 nm,λ_(sec.)=499 nm, λ_(sec.)=534 nm.

Example 8-5% 1,1′-Biphenyl Cross-Linked Polyfluorene NanoparticlesSynthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmop and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmol),3,3′,5,5′-tetrabromo-1,1′-biphenyl (9.4 mg, 20 μmol),tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkylight green solution. DLS (water): Z-average=110 nm, Pdl=0.134, D_(n)=61nm and SD=21.7 nm. UV-Vis Abs. (water): λ_(max)=378 nm, λ_(sec.)=432 nm,λ_(onset)=451 nm. UV-Vis PL (water): λ_(max)=438 nm, λ_(sec.)=466 nm,λ_(sec.)=497 nm, λ_(sec.)=534 nm.

Example 9-5% 9,9′-(1,3-Propanediyl)bis[9-octyl-9H-fluorene] Cross-LinkedPolyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmol),9,9′-(1,3-propyldiyl)bis[2,7-dibromo-9H-Fluorene-9-octyl] (18.3 mg, 20μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkylight green solution. DLS (water): Z-average=118 nm, Pdl=0.133,D_(n)=71.7 nm and SD=24.6 nm. UV-Vis Abs. (water): λ_(max)=383 nm,λ_(sec.)=433 nm, λ_(onset)=451 nm. UV-Vis PL (water): λ_(max)=439 nm,λ_(sec.)=466 nm, λ_(sec.)=498 nm, λ_(sec.)=535 nm.

Example 10-5% 5′-Phenyl-1,1′:3′,1″-terphenyls Cross-Linked PolyfluoreneNanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (76.8 mg, 140 μmol),3,3″,5,5″-tetrabromo-5′-(3,5-dibromophenyl)-1,1′:3′,1″-terphenyl (15.6mg, 20 μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkylight green solution. DLS (water): Z-average=108 nm, Pdl=0.148, D_(n)=66nm and SD=22.5 nm. UV-Vis Abs. (water): λ_(max)=380 nm, λ_(sec.)=433 nm,λ_(onset)=452 nm. UV-Vis PL (water): λ_(max)=439 nm, λ_(sec.)=467 nm,λ_(sec.)=499 nm, λ_(sec.)=535 nm.

Example 11-5% 2,1,3-Benzothiadiazole, 35% 9,9-Di(undecanoicacid)fluorene and 5% 9,9′-Spirobifluorene Cross-Linked PolyfluorenesNanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (1080 μL, 1080 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 2,7-dibromo-9,9-di(undecanoic acid)fluorene (96.9 mg, 140μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol),4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol)tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 20hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL with deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkydark green solution. DLS (water): Z-average=79.0 nm, Pdl=0.117,D_(n)=52.4 nm and SD=15.3 nm. UV-Vis Abs. (water): λ_(max)=380 nm,Δ_(sec.)=450 nm, λ_(onset)=520 nm. UV-Vis PL (water): λ_(max)=535 nm,λ_(sec.)=424 nm.

Example 12-40% Di(t-butyl hexanoate)fluorene and 5% 9,9′-SpirobifluoreneCross-Linked Polyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctyl-9H-fluorene-2,7-diboronic acid bis(pinacol) ester (128.5 mg,200 μmol), 2,7-dibromo-9,9-di(t-butyl hexanoate)fluorene (106.3 mg, 160μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol),tetrakis (triphenylphosphine)palladium(0) (5.8 mg, 5 μmol) andhexadecane (171 μL, 585 μmol). The vial was transferred to an argonfilled glovebox, sealed with a rubber septum and removed. Toluene (2.19mL) was added to the vial and the suspension sonicated until ahomogenous solution was achieved. The initial aqueous solution wascooled to 0° C. in an ice bath, the ultrasonic probe inserted and thetoluene solution injected rapidly into the water. The solution wasultrasonicated for 1 minute, stirred for 30 seconds and ultrasonicatedfor 1 further minute. The Schlenk tube was sealed, placed in a preheatedoil bath at 72° C. and stirred for 20 hours. The Schlenk was opened anda stream of nitrogen gas passed over the emulsion at 50° C., withstirring. The emulsion was cooled to room temperature, the volumeincreased to 23.0 mL with deionised water and filtered through a glasswool plug. The emulsion was obtained as a milky light green solution.DLS (water): Z-average=129 nm, Pdl=0.226, D_(n)=64 nm and SD=23.4 nm.UV-Vis Abs. (water): λ_(max)=384 nm, λ_(onset)=441 nm. UV-Vis PL(water): λ_(max)=430 nm, λ_(sec.)=453 nm, λ_(sec.)=484 nm.

Example 13-5% 4,7-Bis(4-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazoleand 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene NanoparticlesSynthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (76.8 mg, 140 μmol),2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol),4,7-bis(5-bromo-4-hexyl-2-thienyl)-2,1,3-benzothiadiazole (12.5 mg, 20μmop, tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 20hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL with deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkybright red solution. DLS (water): Z-average=105 nm, Pdl=0.125,D_(n)=64.4 nm and SD=20.8 nm. UV-Vis Abs. (water): λ_(max)=382 nm,λ_(sec.)=433 nm, λ_(sec.)=514 nm, λ_(onset)=620 nm. UV-Vis PL (water):λ_(max)=621 nm, λ_(sec.)=437 nm.

Example 14-10% 4,7-Bis(4-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazoleand 5% 9,9′-Spirobifluorene Cross-Linked Polyfluorene NanoparticlesSynthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (65.8 mg, 120 μmol),2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol), (25.1 mg,40 μmop, tris(dibenzylideneacetone) dipalladium(0) (4.6 mg, 5 μmop,tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmop. The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 20hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL with deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkybright red solution. DLS (water): Z-average=130 nm, Pdl=0.264,D_(n)=58.4 nm and SD=20.9 nm. UV-Vis Abs. (water): λ_(max)=382 nm,λ_(sec.)=432 nm, λ_(sec.)=515 nm, λ_(onset)=623 nm. UV-Vis PL (water):λ_(max)=625 nm.

Example 15-2% 9,9-Di(undecanoic acid)fluorene, 5%2,1,3-Benzothiadiazole, 33% Di(hex-5-en-1-yl)fluorene and 5%9,9′-Spirobifluorene Cross-Linked Polyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (816 μL, 816 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmop, 2,7-dibromo-9,9-di(undecanoic acid)fluorene (5.5 mg, 8μmol), 2,2′,7,7′-tetrabromo-9,9′-spirobifluorene (12.6 mg, 20 μmol),4,7-dibromobenzo[c]-1,2,5-thiadiazole (5.9 mg, 20 μmol),2,7-dibromo-9,9-di(hex-5-en-1-yl)fluorene (64.5 mg, 132 μmop,tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmop,tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 20hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL with deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkydark green solution. DLS (water): Z-average=101 nm, Pdl=0.166,D_(n)=55.1 nm and SD=18.1 nm. UV-Vis Abs. (water): λ_(max)=381 nm,λ_(sec.)=453 nm, λ_(onset)=522 nm. UV-Vis PL (water): λ_(max)=530 nm.

Example 16—CL-F8T2 CPNs Synthesis

In a Schlenk tube, sodium dodecyl sulfate (50 mg) was dissolved indeionised water (10 mL) under argon. The solution was degassed bybubbling with argon for 30 minutes. In a separate vial, monomer A (58.6mg, 9.12×10⁻² mmol), monomer B, monomer C (see amounts in Table 1),monomer D (5.8 mg, 9.12×10⁻³ mmol),tris(dibenzylideneacetone)dipalladium(0) (0.9 mg, 0.98×10⁻³ mmol) andtri(o-tolyl)phosphine (1.2 mg, 3.9×10⁻³ mmol) were dissolved in toluene(1 mL). Hexadecane was added (78 μL) and the mixture was degassed bybubbling with argon for 5 min. After this time, the monomer mixture wasthen injected to the SDS solution. To promote the miniemulsion, theSchlenk tube was taken to an ice bath and the mixture was sonicatedusing an ultrasonicator fitted with microtip (Cole Parmer 750 Wultrasonicator, 22% amplitude) for 2 minutes. The tube was resealed andthen heated up to 72° C. Once reached this temperature, an aqueoussolution of sodium hydroxide 1M (365 μL) was added and the reactionmixture was stirred for 16 h. After cooling down to room temperature,the Schlenk tube was opened and the mixture was stirred for 5 h toremove the residual toluene. To remove SDS, 400 μL of the resultingminiemulsion was diluted with 1.6 mL of deionised water and AmberliteXAD-2 (20 mg) previously washed with water (2×2 mL) was added. Themixture was stirred for 2 hours at room temperature and the AmberliteXAD-2 was removed. Treatment with Amberlite XAD-2 was repeated until themixture was shaken and no foam formation was longer observed.

Table 15 below shows the amount of monomers B and C used. Table 16 belowshows the particle size of the CL-F8T2 CPNs. Table 17 shows the opticalproperties of CL-F8T2 CPNs in water & THF.

TABLE 15 Initial loading of monomers B and C in CL-F8T2 CPNs Monomer CMonomer B Monomer C Polymer (% mol) (mass, moles) (mass, moles)CL-F8T2/20 20 20 mg 11.8 mg (3.65 × 10⁻² mmol) (3.65 × 10⁻³ mmol)CL-F8T2/30 30 10 mg 17.8 mg (1.82 × 10⁻² mmol) (5.48 × 10⁻² mmol)

TABLE 16 Particle size of CL-F8T2 CPNs in water & THF Water THF d_(z)D_(Num) d_(z) D_(Num) Polymer (nm) PdI (nm) (nm) PdI (nm) CL-F8T2/20 1050.158 64 124 0.212 62 CL-F8T2/30 103 0.178 53 120 0.223 63

TABLE 17 optical properties of CL-F8T2 CPNs in water & THF Water THFAbsorption Fluorescence Absorption Fluorescence Polymer λ_(max) (nm)λ_(max) (nm) λ_(max) (nm) λ_(max) (nm) CL-F8T2/20 386 554 394 525CL-F8T2/30 431 541 438 498

Example 17—5% N,N,N′N′-Tetraphenylbenzidine Cross-Linked PolyfluoreneNanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmop and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmop, 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmol),N⁴,N⁴,N^(4′),N^(4′)-tetrakis(4-bromophenyl)-[1,1-biphenyl]-4,4′-diamine(16.1 mg, 20 μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5μmol), tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL,585 μmol). The vial was transferred to an argon filled glovebox, sealedwith a rubber septum and removed. Toluene (2.19 mL) was added to thevial and the suspension sonicated until a homogenous solution wasachieved. The initial aqueous solution was cooled to 0° C. in an icebath, the ultrasonic probe inserted and the toluene solution injectedrapidly into the water. The solution was ultrasonicated for 1 minute,stirred for 30 seconds and ultrasonicated for 1 further minute. TheSchlenk tube was sealed, placed in a preheated oil bath at 50° C. andstirred for 16 hours. The Schlenk was opened and a stream of nitrogengas passed over the emulsion at 50° C., with stirring. The emulsion wascooled to room temperature, the volume increased to 23.0 mL usingdeionised water and filtered through a glass wool plug. The emulsion wasobtained as a milky light green solution. DLS (water): Z-average=112 nm,Pdl=0.150, D_(n)=72.5 nm and SD=22.3 nm. UV-Vis Abs. (water):λ_(max)=384 nm, λ_(sec.)=433 nm, λ_(onset)=452 nm. UV-Vis PL (water):λ_(max)=438 nm, λ_(sec.)=467 nm, λ_(sec)=496 nm, λ_(sec.).=535 nm.

Example 18-5% Pyrene Cross-Linked Polyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmop and 1M aqueous sodium hydroxide (800 μL, 800 μmop. Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctyl-9H-fluorene-2,7-diboronic acid bis(pinacol) ester (128.5 mg,200 μmop, 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmop,1,3,6,8-tetrabromopyrene (10.4 mg, 20 μmol),tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmop and hexadecane (171 μL, 585 μmop.The vial was transferred to an argon filled glovebox, sealed with arubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 72° C. and stirred for 20hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL with deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkylight green solution. DLS (water): Z-average=103 nm, Pdl=0.141,D_(n)=71.5 nm and SD=21.8 nm. UV-Vis Abs. (water): λ_(max)=376 nm,λ_(seC.)=432 nm, λ_(onset)=452 nm. UV-Vis PL (water): λ_(max)=439 nm,λ_(sec)=466 nm, λ_(sec.)=498 nm, λ_(sec.)=532 nm.

Example 19-5% 5,10,15,20-tetrakis(4-bromophenyl)-21H,23H-porphine (Zinc)Cross-Linked Polyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmol) and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (87.8 mg, 160 μmol),5,10,15,20-tetrakis(4-bromophenyl)-21H,23H-porphine (zinc) (19.9 mg, 20μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a darkgreen solution. DLS (water): Z-average=95.0 nm, Pdl=0.135, D_(n)=64.1 nmand SD=19.7 nm. UV-Vis Abs. (water): λ_(max)=380 nm, λ_(sec.)=396 nm,λ_(sec.)=433 nm, λ_(sec.)=550 nm, λ_(sec.)=596 nm, λ_(onset)=625 nm.UV-Vis PL (water): λ_(max)=440 nm, λ_(sec.)=466 nm, λ_(sec.)=498 nm,λ_(sec.)=532 nm, λ_(sec.)=605 nm, λ_(sec.)=650 nm.

Example 20-5% 5,10,15,20-Tetraphenyl-21H,23H-porphine (Zinc)Cross-Linked Polyfluorene Nanoparticles Synthesis

In a Schlenk tube was added water (22.0 mL), sodium dodecyl sulfate (110mg, 382 μmop and 1M aqueous sodium hydroxide (800 μL, 800 μmol). Thesolution was purged with argon for 2 hours. In a vial was weighed9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester (111.7mg, 200 μmol), 9,9-dioctyl-2,7-dibromofluorene (65.8 mg, 120 μmol),5,10,15,20-tetrakis(3,5-dibromophenyl)-21H,23H-porphine (zinc) (26.2 mg,20 μmol), tris(dibenzylideneacetone)dipalladium(0) (4.6 mg, 5 μmol),tri(o-tolyl)phosphine (9.1 mg, 30 μmol) and hexadecane (171 μL, 585μmol). The vial was transferred to an argon filled glovebox, sealed witha rubber septum and removed. Toluene (2.19 mL) was added to the vial andthe suspension sonicated until a homogenous solution was achieved. Theinitial aqueous solution was cooled to 0° C. in an ice bath, theultrasonic probe inserted and the toluene solution injected rapidly intothe water. The solution was ultrasonicated for 1 minute, stirred for 30seconds and ultrasonicated for 1 further minute. The Schlenk tube wassealed, placed in a preheated oil bath at 50° C. and stirred for 16hours. The Schlenk was opened and a stream of nitrogen gas passed overthe emulsion at 50° C., with stirring. The emulsion was cooled to roomtemperature, the volume increased to 23.0 mL using deionised water andfiltered through a glass wool plug. The emulsion was obtained as a milkydark green solution. DLS (water): Z-average=98.4 nm, Pdl=0.151,D_(n)=59.9 nm and SD=19.4 nm. UV-Vis Abs. (water): λ_(max)=377 nm,λ_(sec.)=432 nm, λ_(onset)=451 nm. UV-Vis PL (water): λ_(max)=439 nm,λ_(sec.)=466 nm, λ_(sec.)=499 nm, λ_(sec.)=534 nm, λ_(sec.)=596 nm,λ_(sec.)=644 nm.

While specific embodiments of the invention have been described hereinfor the purpose of reference and illustration, various modificationswill be apparent to a person skilled in the art without departing fromthe scope of the invention as defined by the appended claims.

1.-26. (canceled)
 27. A nanoparticle composition comprising a pluralityof nanoparticles formed from a π-conjugated cross-linked polymer, theπ-conjugated cross-linked polymer comprising a) 80-99.9 mol. % ofπ-conjugated monomers, and b) 0.1-20 mol. % of a cross-linker having theformula I shown below:

wherein Z₁ and Z₂ are monomeric moieties, Y is absent, a bond, or alinking group; and wherein the π-conjugated monomers comprise a moietyhaving the formula IV shown below:

wherein R₁ and R₂ are each independently a group:—X-Q wherein X is selected from the group consisting of (1-30C)alkylene,(2-30C)alkenylene, (2-30C)alkynylene, —[O—(CH₂)₂]_(n)—,—(CH₂)_(m)(CF₂)_(n)—, and —[O—Si(R_(z))₂]_(n)—, wherein R_(z) is(1-4C)alkyl, n is 1 to 30, and m is 0 to 30); and Q is a terminal groupselected from hydroxyl, carboxyl, amino, —C═CH₂, —C≡CH, —SH, -biotin,-streptavidin and epoxy.
 28. The nanoparticle composition of claim 27,wherein X is (1-30C)alkylene and Q is carboxyl or amino.
 29. Thenanoparticle composition of claim 27, wherein the π-conjugated monomerscomprise a moiety having the formula:


30. The nanoparticle composition of claim 27, wherein the π-conjugatedmonomers further comprise one or more of the following moieties:

wherein R₃ and R₄ are each independently a group:—X¹-Q¹ wherein X¹ is selected from the group consisting of(1-30C)alkylene, (2-30C)alkenylene, (2-30C)alkynylene, —[(CH₂)₂—O]_(n)—,—[O—(CH₂)₂]_(n)—, —(CH₂)_(m)(CF₂)_(n)—, and —[O—Si(R_(z))₂]_(n)—,wherein R_(z) is (1-4C)alkyl, n is 1 to 30, and m is 0 to 30); and Q¹ isa terminal group selected from hydrogen, methyl, hydroxyl, carboxyl,(1-4C)alkoxycarbonyl, amino, —C═CH₂, —C≡CH, —SH, -biotin, -streptavidin,—CF₃, and a polymerisable group selected from acrylates, epoxy andstyrene; M is a metal selected from Ir, Pt, Rh, Re, Ru, Os, Cr, Cu, Pdand Au; L is a ligand independently selected from the group consistingof halo, (1-30C)hydrocarbyl optionally comprising one or moreheteroatoms selected from N, O, S, Si or P, or an aryl or heteroarylgroup optionally substituted with one or more substituents selected from(1-4C)alkyl, halo, aryl or heteroaryl; and p is 1 to
 4. 31. Thenanoparticle composition of claim 30, wherein X¹ is selected from thegroup consisting of (1-20C)alkylene, —[(CH₂)₂—O]_(n)— or—[O—(CH₂)₂]_(n)— (wherein n is 1 to 20); Q¹ is a terminal group selectedfrom hydrogen, methyl, (1-2C)alkoxycarbonyl and hydroxyl; M is Ir; L isa ligand independently selected from the group consisting of aryl orheteroaryl, optionally substituted with one or more substituentsselected from aryl or heteroaryl; and p is 1 to
 2. 32. The nanoparticlecomposition of claim 30, wherein X¹ is selected from the groupconsisting of (4-12C)alkylene or —[(CH₂)₂—O]_(n)— (wherein n is 1 to15); Q¹ is a terminal group selected from hydrogen, (1-2C)alkoxycarbonyland methyl; M is Ir; L is a ligand independently selected from the groupconsisting of phenyl or 6-membered heteroaryl, optionally substitutedwith one or more substituents selected from phenyl or 6-memberedheteroaryl; and p is 1 to
 2. 33. The nanoparticle composition of claim30, wherein the π-conjugated monomers further comprise one or more ofthe following moieties:


34. The nanoparticle composition of claim 27, wherein the cross-linkerhas the formula II shown below:

wherein Y is absent, a bond, or a linking group.
 35. The nanoparticlecomposition of claim 34, wherein the cross linker has the formula IIIshown below:


36. The nanoparticle composition of claim 34, wherein the cross-linkerhas the following structure:


37. A method of forming a nanoparticle composition as claimed in claim27, the method comprising the step of forming the nanoparticles byemulsion polymerisation, miniemulsion polymerisation or dispersionpolymerisation techniques to provide an aqueous suspension ofnanoparticles.
 38. The method of claim 37, wherein the nanoparticles areformed by a cross-coupling polymerisation reaction.
 39. The method ofclaim 38, wherein the polymerisation reaction is a Suziki reaction. 40.The method of claim 38, wherein the polymerisation reaction is a Stillereaction.
 41. The method of claim 38, further comprising the step ofpurifying the aqueous suspension of nanoparticles.
 42. The method ofclaim 41, wherein the aqueous suspension of nanoparticles is purified bycontacting the aqueous suspension of nanoparticles with at least oneorganic solvent.
 43. The method of claim 42, wherein the at least oneorganic solvent is selected from the group consisting of polar andnon-polar solvents.
 44. The method of claim 43, wherein the at least oneorganic solvent is methanol.
 45. Use of a nanoparticle composition asdefined in claim 27 in one or more applications selected from the groupconsisting of biological or non-biological imaging or sensing,down-conversion of LED light, anti-counterfeit encoding, displays,cell-sorting/flow cytometry, long-term cell tracking, and flowvisualisation.
 46. A nanoparticle dispersion comprising a nanoparticlecomposition as claimed in claim 27 dispersed throughout a dispersingmedium.